Fat Content Determination Methods Teresa McConville Chem 311 Dr. Weisshaar
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1 Fat Content Determination Methods Teresa McConville Chem 311 Dr. Weisshaar The methods used in determination of fat content in foods are as varied as the sample matrices. This is an overview of a few methods applicable or adaptable to a wide variety of foods. The various fat determination methods lead to different definitions of total fat content. For the purpose of labeling, the FDA has defined total fat as the fatty acids obtained from a total lipid extract of the food matrix expressed as triglycerides. 1 These methods of extraction and/or detection may or may not adhere strictly to this definition, and this issue may need to be investigated further depending on the requirements of the determination. Infrared Spectrometry 2 Method In this method the fat is extracted from a homogeneous sample by a chloroform-methanol solvent containing an internal standard. The internal standard found to be most suitable for this procedure was a low viscosity apolar methylsilicone oil (MS 550). The extraction is then analyzed by infrared spectrometry, where the carbonyl ester band of the fat is at 5.72 µm and the internal standard of choice is at 7.88 µm. The areas under the two bands are determined by the triangulation method, or by integration with the IR spectrometer if available. The band area ratio (esterified fatty acids/internal standard) and a calibration curve of weight of fat versus the band area ratio are used to determine the fat content. The calibration curves are prepared by using this same procedure to analyze samples of pure reference fat in the range including the expected fat -1-
2 content of the analyte. Figures of Merit The accuracy and precision of this method are comparable to reference methods which included Rose-Gottlieb for ice cream and liquid egg, Gerber for milk, and Werner-Schmid for other samples. The accuracy in 15 of 20 sample products was not significantly different at the 5% level. For the 5 samples, the fat content was as much as 17% different. Precision varied depending on the food analyzed. The average absolute standard deviation for all products was 0.22 for the reference method and 0.28 for the IR method. Although the limits of detection was not specifically addressed, this study was conducted on samples ranging from 3.3% to 32.54% fat content. IR analysis showed good comparison with standard reference methods in most samples. Observed variations related to food matrix rather than percent fat content. The sensitivity of this method is quite good as the slope of the calibration curves range from 60 to 80 mg fat. The calibration curve is the graph of milligrams of fat versus the band area ratio. The selectivity of this method is better than the reference methods. While the reference methods typically estimate all lipid material in a product, this method determines only the total esterified fatty acids resulting in a lower fat content determination in many products. This agrees with the FDA lableing requirement. For linear dynamic range considerations, the calibration curves demonstrate very good linearity in the band area ratio range of , and deviate only at band area ratios exceeding
3 Overall this method provides a rapid method of fat determination which shows good accuracy and precision depending on the analyte. All the figures of merit are acceptable. If the IR instrument does not have integration capabilities, the tedious triangulation method could be prohibitively slow. Although the calibration curve in some cases depends on the type of food analyzed, most foods can be analyzed with a composite calibration curve. Caviezel Method 3 Method The homogenized sample and the internal standard (IS, tridecanoic acid) are added to the n-butyl alcohol solvent. Potassium hydroxide is used to saponify and extract the fats simultaneously. An acidic aqueous solution is added to convert the fatty acids salts to fatty acids, producing a two phase system where the fats and internal standard are contained in the top layer. This is injected into the fat determination system where it is separated by gas chromatography. The peak areas of the IS and fatty acids are used to determine the fat content which is then converted to triglyceride content with a predetermined factor. The fat determination unit is a commercial instrument which is based on gas chromatography and is geared specifically to this analysis method. This company also produces mixers and extraction units which are designed for this analysis. Figures of Merit The accuracy of this method is very good. A regression of the measured fat content versus the certified fat content of several standard reference materials (SRMs, pork meat, baby food powder, rye flour, and chocolate) resulted in a slope of and R squared of
4 demonstrating a strong correlation. Reproducibility and repeatability were tested with nine types of samples with fat contents ranging from 1 to 79%, taking ten replicates on ten different days. The repeatability relative standard deviations values, RSD r were 0.47 to 3.59% while the reproducibility relative standard deviation values, RSD R, ranged from 0.85 to 9.52%. The recovery from spiked samples averaged 97.2% and yielded a linear regression with R squared The concentration range studied was 0.3 to 100% and the regression of measured versus added fat was linear with R squared of Limits of detection showed 4.3 mg absolute fat in 5 g corresponding to 0.1% fat by the AOAC and ACS definition of limit of detections as blank + 3 standard deviations. According the ACS definition of limit of quantitation as blank signal + 10 standard deviations, this method gives 8.2 mg absolute fat or 0.2% fat content. The laboratories in this study described this method as well documented, easier to follow and shorter than comparable methods. Other comments included that this method involved easier sample handling, and that the instrument was easy to operate. The cost of the gas chromatography based detection unit was not quoted. Simplified Gravimetric after Chloroform-Methanol Extraction 4 Method Samples of 1-5 g are massed into a polypropylene centrifuge bottle. Sodium acetate is added so that the total volume of solution is 32 ml. Aliquots of methanol and chloroform are -4-
5 added, the bottles capped and shaken for 2 hours. Another aliquot of chloroform is added, and the bottles shaken again for 30 minutes. Finally, an aliquot of water is added, and the bottles are shaken a final time for 30 min. Centrifuge tubes which had been dried and massed are used to hold 20 ml aliquots of the chloroform layer. These are then centrifuged for 10 min, and allowed to set in 25-C water bath for 15 minutes. The samples are then evaporated to dryness under a nitrogen blanket, heated in a drying oven for 30 minutes, and cooled in a dessicooler for at least 30 minutes. Finally, they are massed and the total lipid content determined by: total lipid (g/100 g wet weight) = (W 2 -W 1 )*V c *100/(V A *SW) where W 2 is the weight of glass tube and dried extract (g), W 1 is the weight of empty dried glass tube (g), V c is the total volume of chloroform (ml), V A is the volume of extract dried (ml), and SW is the weight of food sample assayed (g). Figures of merit The accuracy of the method was determined by results for NIST Standard reference material (SRM) The mean determined was 20.9 ± 0.52 g/100g dry wt., well within the certified range of 20.6 ± 2 g/100g dry weight. Recovery was essentially quantitative in spiked samples of typical foods (3.2% by weight total lipid) to which ranges of 0.1 to 1.0 g fat was added to 5 g aliquots. The concentration range is limited to 3 to 30% total weight of fat. The precision was determined with 608 samples with average total lipid content from 2.4 to 8.7 g / 100 g wet weight. The within sample covariation was 0 to 9%. The selectivity can be a concern as the chloroform-methanol extraction may overestimate fat content because it extracts lipids other than fatty acids. On the other hand, in may -5-
6 underestimate fat content because low molecular fatty acids might not be extracted. This is a modification of AOAC and involves less analyst time, less solvent loss and the Clarase reagent is not needed. It is more applicable to batch analysis and simpler than the AOAC method, yet the unfavorable halogenated hydrocarbon solvent is still required. Gravimetric analyses are severely dependant on mass and volume accuracy. This factor leaves this method generally unfavorable. Supercritical CO 2 Extraction 5 Method The finely ground sample is massed and placed in an extraction cartridge and mixed with an equal amount of ISCO (ISCO Inc, Lincoln, NE) wet support. SFC/SFE grade CO 2 is used with a commercial instrument at pressures up to 10,000 psi, temperatures of about 80-C and approximately a 4 ml/min flow of liquid CO 2 for about 30 minutes to extract fat content. The extraction parameters vary depending on the sample matrix. After extraction the collection vial is heated for 20 minutes at 120-C. The collection vial is massed before and after extraction. The variable conditions include extractor temperature, pressure, modifiers of ethanol or ethanol and water mixture, restrictor temperature, flow rate, and extraction time. Figures of Merit Standard figures of merit were not reported in this article. SFE was compared to standard reference methods for six different categories of foods, including dairy, chocolate, and gravies. The reference methods were International Dairy Federation, Weibull, Twisselmann, and several IUPAC methods. In all cases SFE showed bias in reporting a lower fat content than the -6-
7 reference. The repeatability and reproducibility in most cases were good, however not quite as good as the reference methods. The concentration range was from less than 1% to 35% fat content. Overall this method provides a rapid, fully automated determination. There is a significant reduction in the used of solvents and avoids the use of the halogenated hydrocarbons, there is low thermal damage to the sample and there exists the possibility of selectively isolating components. The high cost and complex equipment are the main disadvantages. There is also incomplete recovery for some products, as well as the fact that the method and conditions must be developed for different sample matrices. Conclusions The methods reviewed all had acceptable figures of merit. Supercritical CO 2 extraction is the simplest and quickest method, however the capital equipment cost may be prohibitive. The Caviezel method is also rapid and largely automated, but again the cost of special equipment may be undesirable. If a large capital expenditure and method development expense is acceptable, the supercritical CO 2 method is advantageous in the elimination of the use of halogenated hydrocarbons. The gravimetric method is unfavorable due to the errors introduceable in the mass and volume measurements. Although the IR method uses the unfavorable halogenated hydrocarbon, it has the advantage of some automation with the use of nonspecialized equipment. Assuming an IR spectrometer with integration capabilities is available, this method would be preferred. -7-
8 References: 1 Ngeh-Ngwainbi, J.; Lane R.H. Perspectives on Fat Content. Cereal Foods World. 1994, 39(2), Cronin, D.A.; McKenzie, K. A Rapid Method for the Determination of Fat in Foodstuffs by Infrared Spectrometry. Food Chem. 1990, 35, Pendl, Roger; Bauer, Marcel; Caviezel, Rafael; Schulthess, Peter. Determination of Total Fat in Foods and Feeds by the Caviezel Method, Based on a Gas Chromatographic Technique. J. AOAC Int. 1998, 81(4), Phillips, Katherine M. Simplified Gravimetric Determination of Total Fat in Food Composites After Chloroform-Methanol Extraction. JAOCS. 1997, 74(2), Dionisi, F.; Hug, B.; Aeschlimann, J.M.; Houllemnar, A. Supercritical CO 2 Extraction for Total Fat Analysis of Food Products. Journal of Food Science. 1999, 64(4), King, J.W. et al. Extraction of Fat from Ground Beef for Nutrient Analysis Using Analytical Supercritical Fluid Extraction. J. Agric. Food Chem. 1996, 44, Szakas, Tom. Just the Fats, Ma am: A Methods Overview of Total Fat Determination. Food Testing & Analysis. 1999, 5(1),
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